The broad aim of this research is to study the mechanisms which regulate the synthesis of glucose in mammalian liver. We will be especially interested in the role of mitochondrial P-enolpyruvate carboxykinase in species such as the guinea pig, rabbit and human, and its role in gluconeogenesis. We will extend our previous findings which indicate that ammonia may act as a normal biological oxidant which alters the effect of fatty acid oxidation on gluconeogenesis in these species. Differences in role of gluconeogenesis and ketogenesis between rat and guinea pig livers in response to changes in oxygen tension will be studied, with particular emphasis on the role of the mitochondrial oxidation-reduction state on this process. The factors responsible for the maintenance of an extremely reduced cytosol oxidation-reduction state, as noted in species having substantial mitochondrial P-enolpyruvate carboxykinase (guinea pig, rabbit) will be investigated using liver perfusion and isolated liver cell preparations. Both the mitochondrial and cytosol forms of P-enolpyruvate carboxykinase from guinea pig liver and the cytosol form of the enzyme from rat liver will be isolated and their kinetic properties studied. The major emphasis of this work will be the determination of the order of substrate binding, and various kinetic constants to relate to known physiological rates of P-enolpyruvate formation in the liver. The specific metal ion which binds to P-enolpyruvate carboxykinase and is sensitive to 3-mercaptopicolinate inhibition will be determined.